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Despite the ecologic and ornamental potential of southeastern U.S. native Spigelia, little is known about the intraspecific or the interpopulation genetic variation. The southeastern U.S. native Spigelia habitat is becoming more and more fragmented as a result of human activity, making it imperative to gain an understanding of natural genetic variation among and within species and populations for the purpose of obtaining variability for plant breeding and preserve the genetic variability in Spigelia. Therefore, the objective of this study was to use amplified fragment length polymorphism analysis to determine interspecific and intraspecific genetic variation and to evaluate gene flow. Thirteen populations of two species of native Spigelia, S. marilandica (SM), S. gentianoides var. gentianoides (SGG), and S. gentianoides var. alabamensis (SGA), were analyzed using four primer pairs that amplified a total of 269 bands. Based on analysis of molecular variance and estimates of Nei’s coefficients of gene diversity (percentage of polymorphic loci, average genetic diversity within populations, average genetic diversity within species, and proportion of species genetic diversity attributed to among population variation), the majority of variation found in Spigelia occurs within populations. Both among-species and among-population variation was low, likely the effect of common ancestry as well as relatively frequent introgression among individuals (and populations) of Spigelia. When all individuals were evaluated using Nei’s unbiased genetic distances and viewed as a unweighted pair group method with arithmetic mean phenogram, three main groups were shown, one with two samples of SGG from one population, one with 13 individuals from both SGG populations used in this study, and one with all of the SM, SGA, and remaining SGG individuals. Further evaluation using STRUCTURE software showed introgression between populations and species, although all allele clusters have not entirely introgressed into all populations. The significance of these results is discussed in relation to breeding in Spigelia. Spigelia (Loganiaceae) is a genus of 50 species that ranges from the southeastern United States to Central America and south to temperate areas of South America. Only five species are considered endemic to the United States and two of these are indigenous to the southeastern United States: S. marilandica and the federally endangered Spigelia gentianoides (Gould, 1997). To aid recovery of S. gentianoides, research on the genetic variability of the natural populations of this species has been encouraged (Negron-Ortiz, 2012). Furthermore, these species are of interest to plant breeders as a result of their ornamental potential. Therefore, for purposes of obtaining variability for plant breeding and to preserve the genetic variability in Spigelia, evaluation of the genetic variability within and among the species is essential. Observation of physical traits of Spigelia species has shown morphological variation among species, populations within a species, and between plants of the same population. Although these morphological differences have been observed, the degree of genetic difference in these two species has yet to be determined. Spigelia gentianoides (SG) is an upright, perennial herbaceous species that has two disjunct botanical varieties, and both are considered endangered (Gould, 1997). Threats to this species include loss or alteration of habitat, lack of natural disturbance regimes, unprotected populations on private lands, and competition from invasive species (Negron-Ortiz, 2012). Spigelia gentianoides var. alabamensis has pink corollas from 36 to 50 mm long and their lobes reflex (open) at anthesis. Flowering occurs from May to June. This botanical variety is endemic to 17 glades that have developed over an ancient rock formation known as Ketona Dolomite in Bibb County, AL (Negron-Ortiz, 2012). The glades vary in size from 0.1 to 5 ha. Spigelia gentianoides var. gentianoides has pink flowers that are barely opening and not reflexed at anthesis (Table 1). Flowering is from May to July. SGA differs from SGG in that it has longer corollas with broader throats, longer lobes as well as longer sepals (Table 1) (Gould, 1997). SGG is restricted to five locations within three counties in the Florida Panhandle and southeastern Alabama. Gould labeled SGA as a botanical variety as opposed to a species designation (1997) despite listing numerous and distinct trait differences between the two botanical varieties. Subsequent assessment of SG and its two botanical varieties suggests that the two SG botanical varieties warrant specific Received for publication 26 June 2014. Accepted for publication 18 Dec. 2014. This study is a portion of a dissertation submitted by Amanda Hershberger in partial fulfillment of the requirements for a PhD degree. We thank Drs. Noelle Barkley and Zhenbang Chen and Mr. Tyler Eaton for assistance with amplified fragment length polymorphism methodology and data analysis. Corresponding author. E-mail: croback@uga.edu. 120 J. AMER. SOC. HORT. SCI. 140(2):120–128. 2015. rank (Weakley et al., 2011), although additional molecular and morphological studies were suggested. Spigelia marilandica is an upright perennial herbaceous plant growing from 30 to 60 cm high. Corollas are bright red outside and yellow to greenish yellow inside (Table 1). In Kentucky, the flowering period starts in late May through June; occasionally scattered blooms will occur in the fall (Dunwell, 2003). It is thought to be pollinated by ruby-throated hummingbird [Archilochus colubris (Cullina, 2000)] as well as by insects (Affolter, 2005; Rogers, 1988). Distribution is from eastern South Carolina to north–central and northwestern Florida, westward to southeastern Oklahoma and far eastern Texas, and north to Kentucky (Duncan and Duncan, 2005; Gould, 1997). Limited research has been conducted to determine genetic diversity in Spigelia. Gould (1997) used internal transcribed spacer (ITS) sequencing to assess relationships of 14 species of Spigelia, whereas Affolter (2005) used allozymes to evaluate genetic variability in both botanical varieties of SG as well as in SM. A technique that is especially useful to assess genetic diversity within and among species when no sequence information is available, as is the case in Spigelia, is amplified fragment length polymorphism (AFLP) analysis. AFLP technology has been used to characterize genetic diversity within many genera of plants including horticultural crops such as Chrysanthemum sp. (Zhang et al., 2010), sweetpotato [Ipomoea batatas (Cervantes-Flores et al., 2008)], pecan [Carya illinoinensis (Beedanagari et al., 2005)], Rhododendron sp. (Chappell et al., 2008), barberry [Berberis thunbergii (Lubell et al., 2009)], and Lactuca sp. (Koopman et al., 2001). AFLP markers have a very high diversity index (a measure of evaluation efficiency that combines the effective number of alleles identified per locus and the number of polymorphic bands in each assay) (Philips and Vasil, 2001; Russell et al., 1997) and is therefore appropriate for use in the evaluation of genetic variability in Spigelia. We used AFLP technology to determine genetic differences within and among populations of two Spigelia species indigenous to the southeastern United States. Materials and Methods PLANT MATERIAL. Ten wild populations of SM, one of SGA, and two of SGG were used in genetic analysis (Table 2; Fig. 1). Before collection of plant material, the area was surveyed to define the population area. Plants were collected evenly throughout the population and locations of individual plants collected in each population were recorded using global positioning system coordinates. The populations’ size ranged from 14 to 800 and the number of plants evaluated per population ranged from 4 to 11 (Table 2). Approximately 100 mg of immature leaves of each sample were collected onsite, placed on ice in a cooler, and transported to the laboratory for immediate extraction of DNA. DNA EXTRACTION PROTOCOL AND QUANTIFICATION. DNA extraction was carried out using the E.Z.N.A. plant DNA kit (Omega Bio-Tek, Norcross, GA) with slight modifications including the addition of 26 mL b-mercaptoethanol to Step 1, an increase of the incubation time at 65 C for 30 min in Step 2, the use of isopropanol stored at –20 C in Step 4, and the use of 0.9 mL carrier RNA in the initial column step. DNA was tested for quantity and quality (shearing) using a standard agarose gel with the Low DNA Mass Ladder (Invitrogen, Carlsbad, CA). Subsequently, genomic DNA was stored at 4 C until AFLP analysis was performed. Table 2. Locations of Spigelia marilandica (SM), S. gentianoides var. alabamensis (SGA), and S. gentianoides var. gentianoides (SGG) populations collected and grouped by species.

Molecular Genetic Variability of Spigelia marilandica and S. gentianoides